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Anti-inflammatory effect of alpha lipoic acid loaded calcium citrate nanoparticle on
human keratinocyte HaCaT cells
Boonyaras Bukkavesa, M.D., Pol.Maj.Gen.1*
1Police General Hospital, Bangkok, Thailand
Tel.: +(66)8-1837-9459 E-mail: [email protected]
Siwaporn Nilyai, M.Sc.2
2Nanomedicine Research Unit, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Tel.: +(66)8-9517-2320 E-mail: [email protected]
Kanidta Sooklert, Ph. D.2
2Nanomedicine Research Unit, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Tel.: +(66)9-1716-5964 E-mail: [email protected]
Sasin Thamakaison2
2Nanomedicine Research Unit, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Tel.: +(66)6-5378-5692 E-mail: [email protected]
Amornpun Sereemaspun, M.D., Ph.D.2
2Nanomedicine Research Unit, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Tel.: +(66)9-6569-8859 E-mail: [email protected]
*Correspondence: Boonyaras Bukkavesa
1Police General Hospital, 492/1 Rama 1 Road, Rajprasong Intersection, Pathumwan
Bangkok, Thailand.
Tel.: +(66)8-1837-9459
E-mail: [email protected]
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Running title: Lipoic nanoparticle anti-inflammation in keratinocytes
Abstract
Background: Alpha lipoic acid (LA) is potent antioxidant and anti-inflammation natural
compound. To enhance the efficacy and stability of LA, LA-loaded calcium citrate
nanoparticles were developed.
Objective: To investigate the anti-inflammatory effects of LA-loaded calcium citrate
nanoparticles (LA-NPs) in HaCaT keratinocytes.
Methods: HaCaT cells were exposed under two inflammation stimuli conditions, LPS and
Pb(NO3)2, and then were treated with or without LA and LA-NPs for 24 h. Cell viability
and pattern of cell death were evaluated. In addition, the expression levels of inflammatory
cytokines including IL-1β, IL-6, and TNF-α as well as inflammatory mediator COX-2
were determined by real-time PCR.
Results: LA and LA-NPs decreased the percentage of cell death in both LPS and
Pb(NO3)2-induced conditions. In LPS-induced cells, LA and LA-NPs attenuated the fold of
gene expression levels in IL-1β, IL-6, TNF-α, and COX-2. Decreased expression in
proinflammatory cytokines, including IL-1β and IL-6, was also observed in Pb(NO3)2-
induced cells.
Conclusion: Our study demonstrated the anti-inflammatory effects of LA and LA-NPs on
LPS and Pb(NO3)2-induced human keratinocytes.
Keywords: Alpha lipoic acid, calcium citrate nanoparticle, anti-inflammation
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Introduction
Skin, an organ that covers the whole human body, acts as first line of barrier to
prevent the entry of harmful substances and environmental stimuli into the body. The skin
immune response is an importance mechanism to prevent irritation and injury from toxic
or foreign substances such as microorganism infection, UV irradiation, pollutants exposure
(1). Owing to the structure of skin, epidermal keratinocytes plays a crucial role in
receiving environmental signals and acts as initiator of inflammation through the
expression of cytokines and chemokines in activating signaling cascades (2). Although
acute inflammation in skin cells is simply a protective reaction towards foreign objects and
various stresses, the alteration of skin structure or function from excessive and long-term
stress exposure results in chronic inflammation that may lead to development of skin
diseases including cancer and premature aging (3, 4).
Protective and therapeutic skin drugs, including cosmeceuticals in topical
application, are one of the solutions to treating inflammatory skin diseases. Recently,
natural compounds have gained interest over synthetic drugs as alternative therapies
because their lack of side effects. One example of a natural compound with potential
therapeutic properties is α-Lipoic acid (LA), as it shows potent antioxidant effects (5) and
also acts as anti-inflammatory agent under various experimental conditions (6-8). LA plays
the essential role as a coenzyme of multiple enzyme complexes and is involved in cellular
metabolism (9). LA has also been shown to be involved in the regeneration of other
antioxidants such as Vitamin C, Vitamin E (10) as well as glutathione. Furthermore, it has
shown properties as a free radical scavenger and has shown antioxidant activity as a metal
chelator against some heavy metal ions (11). Furthermore, LA’s anti-inflammation
properties were also reported in both in vitro and in vivo models. In in vitro studies, LA
has been shown to attenuate the level of inflammatory cytokines, including TNF-α, IL-1β
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and IL-6 and level of NF-κB, in H2O2-treated fibroblast cells (8) and has also demonstrated
the ability to reduce inflammatory cytokine secretion in human keratinocytes (12). In in
vivo studies, LA has shown an anti-inflammation effect on acute and chronic inflammation
in rats because of its strong anti-oxidative potency (13). Moreover, LA showed anti-
inflammatory and antiapoptotic effects by reducing TNF-α, iNOS, COX-2 and caspase-3
levels in liver tissue rat hepatic injury (14).
Due to LA’s unique characteristics, it has potential to be therapeutically applied in
a wide range of clinical conditions, especially to treat oxidative stress-associated diseases
(15). However, LA still possesses various limitations that inhibits its medical applicability:
this includes its low stability, low cellular uptake, fast biodegradability, and low
penetration capacity in topical administration at the skin (16). This has led several research
groups to focus on enhancing LA’s efficacy and stability through combining it with other
compounds or through chemical modification. In recent years, nanoparticles-based drug
delivery systems have received notable attention because they have the unique capacity in
improve the drug stability and increase treatment efficacy (17). The controlled release
system of nanoparticles-conjugated compounds allow better penetration and controlled
drug release at the target site (17). Among several types of nanoparticles, calcium
nanoparticles is one of the most common inorganic compounds that has high availability,
low cost, low toxicity, and slow biodegradation. With this, it may be assumed that calcium
nanoparticles acts not only to carry drugs in a delivery system but also helps to retain
drugs after administration.
Therefore, calcium citrate nanoparticles (CaCitNPs), another type of calcium
nanoparticle with beneficial properties, were developed in order to enhance LA’s
therapeutic benefits and its control release properties. In this study, LA-loaded calcium
citrate nanoparticles (LA-NPs) were synthesized by our novel method outlined in our
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previous report. Nevertheless, the abilities of LA-NPs still need to be investigated to
confirm that they retain the effective activities of the original LA compound. Previously,
we have reported that LA-NPs demonstrate the cytoprotective property against H2O2-
induced oxidative stress in human keratinocytes (18). However, the anti-inflammatory
properties of modified LA-NPs on cells have yet to be investigated. Thus, this study aims
to investigate the anti-inflammatory response of human keratinocyte (HaCaT) cells that
have been induced by two different types of inflammation trigger agents,
lipopolysaccharide (LPS) and lead (II) nitrate (Pb(NO3)2), to synthesized LA-NPs.
Viability and cell death pattern of HaCaT cells after treatment with LA and LA-NPs under
inflammatory-induced conditions were detected as well as the level of pro-inflammatory
cytokines including interleukine-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and
interleukine-6 (IL-6). The levels of a mediator of inflammation cyclooxygenase-2 (COX-
2) were also determined.
Materials and Methods
LA loaded CaCitNPs (LA-NPs) preparation
Synthesis of LA loaded CaCitNPs had been clearly explained in our previous
study. Briefly, calcium chloride and lipoic acid were mixed together and then trisodium
citrate was added and stirred for 10 min. Distilled water was added to discard large
particles. The suspension was centrifuged in order to collect the precipitate and store it as
stock in powder form. Before usage in the experiment, fresh LA-loaded CaCitNPs were
dissolved in distilled water and sonicated.
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Cell cultures
Human keratinocyte cell line, HaCaT, was purchased from American Type Culture
Collection (ATCC, USA). The cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at
37 °C in 5% CO2 humidified atmosphere.
Viability measurement
HaCaT cells were seeded at density 1x104 cells in 96-well plate and incubated
overnight. The cells were pre-incubated with LPS or Pb(NO3)2 and were then treated with
or without LA and LA-NPs for 24 h. Viability of cells were determined by PrestoBlue™
cell viability reagent (Invitrogen, USA), and cell permeable reasazurin-based solution.
After incubation, 10 µl of PrestoBlue® were added to each well and then the samples were
incubated at 37 °C for 30 min. Fluorescence intensity of viable cells were detected by
Varioskan Flash microplate reader (Thermo Scientific, USA) at 560 nm emission and 590
nm excitation.
Cell death pattern
To determine pattern of cell death after exposure to inflammatory conditions and
treatment with LA and LA-NPs, Annexin V and propidium iodide (PI) staining were used.
HaCaT cells were seeded into 12-well plate at 5x105 cell each well and incubated
overnight. After 24 h of treatment, the cells were collected and resuspended in 1x
phosphate buffer saline (PBS). The pellet of cells after centrifugation were stained with
fluorescein isothiocyanate (FITC)-conjugated Annexin V and PI (BD Biosiciences, USA)
for 15 min at room temperature in dark place. The cells were analyzed by flow cytometry
instrument (Backman Coulter CytoFlex, USA).
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Inflammatory gene expression
After 24 h incubation, the treated cells were harvested for gene expression analysis.
Total RNA was extracted using TRIzol reagents (Invitrogen, USA). Concentration of
isolated RNA of each sample was measured using NanoDrop™ spectrophotometers
(Thermo Scientific), and 500 ng/µl of total RNA was employed in the next step.
Quantitative real-time PCR was performed on ABI step-one plus using SensiFAST™
SYBR® One-Step Kit (Bioline, UK). Total RNA was converted into cDNA and target
gene expression was amplified in one-step Real-Time PCR using the following conditions:
reverse transcription at 45 °C for 10 min, polymerase activation at 95 °C for 2 min and 40
cycles of denaturation and annealing were performed at 95 °C for 5 s and 60 °C for 30 s,
respectively. Sequence of gene-specific primers were as follows: IL-1β forward, 5’-AGA-
TGA-TAA-GCC-CAC-TCT-ACA-G-3’ and reverse, 5’ACA-TTC-AGC-ACA-GGA-
CTC-TC-3’; IL-6 forward, 5’-GTGTGAAAGCAGCAAAGAG-3’ and reverse, 5’-
CTCCAAAAGACCAGTGATG-3’; TNF-α forward, 5’ -
TCCTTCAGACACCCTCAACC-3’ and reverse, 5’-AGGCCCCAGTTTGAATTCTT-3’;
COX-2 forward, 5’-TTCTCCTTGAAAGGACTTATGGGTAA-3’ and reverse, 5’-
AGAACTTGCATTGATGGTGACTGTTT-3’; GAPDH forward, CAT-CAC-CAT-CTT-
CCA-GGA-GCG and reverse, GAG-GGG-CCA-TCC-ACA-GTC-TTC. Melt curve
analysis was performed to confirm the specificity of the amplified product. Fold changes
of gene expression were calculated using comparative Ct method (2-∆∆Ct) and relative
expression of inflammatory genes were calculated using GAPDH as a housekeeping
control.
Statistical analysis
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All data were analyzed using GraphPad Prism and results were expressed as
mean±SD. One-way analysis of variance (ANOVA) was used to detect significant
differences between treatment groups followed by Tukey’s post hoc multiple comparison
test. Results were considered to be significantly different when p values were < 0.05.
Results
Cell viability
To determine the cytotoxicity of the inflammation-inducing agents and the effects
of LA and LA-NPs on keratinocytes under inflammation-triggered condition, cells were
exposed with 10 µg/ml LPS and 100 µg/ml Pb(NO3)2 and were treated with or without LA
and LA-NPs at concentration 5 and 10 µg/ml for 24 h (Fig 1). LPS and Pb(NO3)2 slightly
decreased the percentage of cell viability. However, the cells exposed with LPS and LA at
10 µg/ml showed a significant increase in percentage of viability than LPS treatment
alone. Also, the cells exposed to Pb(NO3)2 with LA at 10 µg/ml and LA-NPs at 5 and 10
µg/ml showed significantly increased percentage of viability compared to Pb(NO3)2 alone.
Cell death pattern
To evaluate the effect of LA and LA-NPs on the cell death pattern of cells treated
with LPS or Pb(NO3)2 , annexin V-FITC/PI staining was performed using flow cytometry
analysis. Cells were induced with 10 µg/ml of LPS (Fig. 2A-B) or 100 µg/ml of Pb(NO3)2
(Fig. 2C-D) and were treated with LA or LA-NPs 10 µg/ml for 24 h. The results showed
both inflammatory-induced agents can induce necrosis in HaCaT cells as 25.23% and
29.3% of total cells in LPS and Pb treatment, respectively. On the other hands, in treatment
with LA and LA-NPs 10 µg/ml with LPS can decrease percentage of necrotic cells into
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11.23 and 12.24, respectively. The same results were found in Pb(NO3)2–induced group as
10.29% and 8.37% of necrotic cells in LA and LA-NPs treatment, respectively.
Gene expression level of inflammatory cytokines
Real-time PCR was performed to detect the mRNA expression level of
inflammatory cytokines, including IL-1β, IL-6, TNF-α, and COX-2, in cells exposed to
LPS or Pb(NO3)2 alone compared to the groups that combined treatment with LA or LA-
NPs. As shown in Fig 3., LPS-induced cells demonstrated significantly increased genes
fold changes of IL-6, TNF-α and COX-2 from the control. IL-1β in LPS-induced cells also
demonstrated a slight increase in expression level. Combined treatment of LPS and LA
attenuated the levels of IL-6, TNF-α and COX-2. LA-NPs with LPS-treated cells showed
suppression of IL-1β, IL-6 and TNF-α levels. However, the expression of all inflammatory
cytokines showed no statistically significant difference between LA and LA-NPs treatment
groups. The same results showed in Pb(NO3)2-treated groups for expression of IL-1β and
IL-6 (Fig. 4 A and B).
Discussion
LA is a natural compound that exhibits various potent antioxidant and anti-
inflammation properties that candidates it to be used as therapeutic drug for several
diseases (19). For skin, drugs in topical application form is effective because it can reach
the target site directly. LA might be developed to potentially be used as anti-inflammatory
drug for skin diseases but its properties of low skin penetration and low bioavailability are
still critical limitations. Our research group has modified the LA drug delivery system
through calcium citrate nanoparticles and have demonstrated in the previous report that
LA-conjugated calcium citrate nanoparticles can better protect the keratinocyte cells from
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oxidative stress induced agents than LA alone (18). In this study, we showed the anti-
inflammatory effect of LA-NPs under LPS- and Pb(NO3)2 induced conditions.
Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-
negative bacteria, has been demonstrated to be a key molecule in triggering an immune
response through the activation proinflammatory cytokines and chemokines (20).
Prolonged exposure to LPS can lead to cell injury and necrosis (21). Previous study has
revealed that LPS can activate proinflammatory cytokines, including IL-1β, IL-6, and
TNF-α, in keratinocyte cells (22). COX-2, a prostaglandin-endoperoxide synthase (PTGS),
is readily induced in response to inflammatory stimuli, including LPS. Since the critical
role of COX-2 in mediating inflammatory processes and involvement in pathogenesis has
been verified in skin model, it is determined as anti-inflammatory-targeted molecule. (23).
Therefore, LPS were used in this study as the stimuli to construct in vitro cell skin
inflammation model. In this study, we found that LA-NPs and LA can inhibit expression
of IL-1β, IL-6, TNF-α, and COX-2 in LPS-induced HaCaT cells along with improve
percentage of cell death. Protective effects of LA in LPS-induced models have been
reported (20, 24, 25). In addition, LA has been shown ability in decrease IL-1β, IL-6,
TNF-α level in H2O2-induced in rat embryonic fibroblast cells (8) and suppress expression
of IL-1β, IL-6, TNF-α, and COX-2 in LPS-induced kidney cells (26). A previous study of
LPS-induced monocytes demonstrated that LA inhibit the effect of LPS by activating the
PI3K/Akt pathway (27). To investigate effect of LA-NPs on other toxic substances, lead
(II) nitrate (Pb(NO3)2) was selected to stimulate an inflammatory response of an
environment pollutant, as the toxic effects of Pb2+ are associated with inflammatory
diseases (28). The results of several studies have clearly confirmed that Pb plays a crucial
role in development of inflammation by acting on the level of gene expression and the
production of proinflammatory proteins (29). In our study, 100 µg/ml of Pb(NO3)2 showed
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an increased percentage of cell death and increased expression levels of IL-1β and IL-6
proinflammatory genes. As expected, LA-NPs and LA can suppress both IL-1β and IL-6
expression and improve the level of viability cells. As a result of this study, LA-NPs have
showed potential in attenuating the expression of proinflammatory cytokines in both LPS
and Pb(NO3)2-induced keratinocyte cells.
LA-NPs and LA demonstrated the same strength of anti-inflammatory effect in
LPS and Pb(NO3)2-induced HaCaT cells. However, because of the drug-delivery
nanoparticle-assembling process, the concentration of LA in LA-NPs was not comparable
with that of LA alone. As the drug loading capacity of LA-NPs limits the amount of LA
encapsulated into calcium citrate, the exact amount of LA in LA-NPs must be less than
weighted. The previous study has been revealed that calcium nanoparticles can improve
the efficiency of drug delivery by enhancing cellular uptake (30). Moreover, drugs or
bioactive proteins in calcium nanoparticles system have reported good sustained-release
performance and high stability (17). Therefore, the equal anti-inflammatory effect of LA
and LA-NPs, which is composed of a smaller amount of LA, may be due to calcium
nanoparticles’ beneficial properties.
In the present study, we demonstrated the anti-inflammatory effect of LA-NPs on
keratinocyte cells via attenuated expression of associated inflammatory cytokines in both
LPS and Pb(NO3)2-induced models. These findings provide support for the effect of
modified LA on inflammatory responses of skin cells. However, the mechanisms of LA in
nanoparticles-based delivery form and the specific properties of calcium nanoparticles that
encourage LA-NP’s effect on keratinocyte cells are still unknown and needs to be clarified
in further research.
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Conclusion
In this study, modified LA-loaded calcium citrate nanoparticles demonstrated an
anti-inflammatory effect that was same as LA in different toxic substance-induced
inflammation conditions in skin cells models. However, the efficacy of controlled release
system of nanoparticles-based should be investigated and stability of LA with carrier must
be verified in future studies.
Fig 1. Effects of LA and LA-NPs on viability in HaCaT cells treated with LPS (A) and
Pb(NO3)2 (B). Cells were treated with 10 and 100 µg/ml LPS and Pb(NO3)2, respectively
and then treated with 5 and 10 µg/ml LA or LA-NPs for 24 h. Fluorescence of viable cells
were detected. Data represent the mean value of triplicate ± SD. ##p < 0.01 versus control
and *p < 0.05 **p < 0.01 ***p < 0.001 versus LPS or Pb.
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Fig 2. Effects of LA and LA-NPs on death pattern of HaCaT cells treated with LPS (A and
B) and Pb(NO3)2 (C and D). Cells were treated with 10 and 100 µg/ml LPS or Pb(NO3)2,
respectively and then treated with 10 µg/ml LA or LA-NPs for 24 h. Cell dead pattern
were detected by annexin V-FITC/PI staining. A) and C) showed scatter plots of annexin
V-FITC/PI staining. B) and D) represented quantitative analysis.
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Fig 3. Effects of LA and LA-NPs on mRNA expression level of pro-inflammatory
cytokine in LPS-induced HaCaT cells. Cells were treated with 10 µg/ml LPS and then
treated with 10 µg/ml LA or LA-NPs for 24 h. The mRNA levels of IL-1β (A), IL-6 (B),
TNF-α (C), and COX-2 (D) were detected by Real-time PCR. Data represent the mean
value of triplicate ± SD. #p < 0.05 versus control and *p < 0.05 versus LPS.
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Fig 4. Effect of LA and LA-NPs on the mRNA level of pro-inflammatory cytokine in
Pb(NO3)2-induced HaCaT cells. Cells were treated with 100 µg/ml Pb(NO3)2 and then
treated with 10 µg/ml LA or LA-NPs for 24 h. The mRNA levels of IL-1β (A), IL-6 (B),
TNF-α (C), and COX-2 (D) were detected by Real-time PCR. Data represent the mean
value of triplicate ± SD. #p < 0.05 versus control. *p < 0.05 and **p < 0.01versus Pb.
Acknowledgments
The authors gratefully acknowledge the Grant to support a research group in the Research Unit Endowment Fund, Chulalongkorn University.
Conflict of Interest
The authors report no conflicts of interest.
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